1. Field of the Invention
This invention relates, generally, to screening assays for diseases and disorders related to cell proliferation. More particularly, it relates to specific antibody-based screening assays for colorectal cancer.
2. Brief Description of the Prior Art
Many common cancers can be treated more successfully when they are detected early, either as precursor lesions or early-stage malignancies, though the choice of markers for early detection is a challenge. The aim is to distinguish normal cells from cancer cells, irrespective of the oncogenes or tumor-suppressor genes that the cancer cells mis-express. Abundant evidence indicates that DNA replication proteins are exceptionally good markers for early detection of many of the common carcinomas and that they can also provide clinically useful prognostic information. The Mcm2 protein is particularly valuable in this role, outperforming both PCNA and Ki67, which are two other replication proteins that have also been used for this purpose (Davies, R., Miller, R., and Coleman, N. (2005) Colorectal cancer screening: Prospects for molecular stool analysis. Nat Rev Cancer 5, 199-209; RG, C.-B., R, G.-G., N, M.-F., and R, B.-M. (2015) Immunoexpression of Ki-67, MCM2, and MCM3 in Ameloblastoma and Ameloblastic Carcinoma and Their Correlations with Clinical and Histopathological Patterns. Dis Markers Epub, 683087; Razavi, S., Jafari, M., Heidarpoor, M., and Khalesi, S. (2015) Minichromosome maintenance-2 (MCM2) expression differentiates oral squamous cell carcinoma from pre-cancerous lesions. Malays J Pathol 37, 253-258; Joshi, S., Watkins, J., Gazinska, P., Brown, J., Gillett, C., Grigoriadis, A., and Pinder, S. (2015) Digital imaging in the immunohistochemical evaluation of the proliferation markers Ki67, MCM2 and Geminin, in early breast cancer, and their putative prognostic value. BMC Cancer 15, 546; Zheng, J. (2015) Diagnostic value of MCM2 immunocytochemical staining in cervical lesions and its relationship with HPV infection. Int J Clin Exp Pathol. 8, 875-880; Hua, C., Zhao, G., Li, Y., and Bie, L. (2014) Minichromosome Maintenance (MCM) Family as potential diagnostic and prognostic tumor markers for human gliomas. BMC Cancer 14, 526).
Dysregulation of DNA replication is fundamental for uncontrolled cellular proliferation, and the clinical targeting of eukaryotic replication factors has seen widespread use in cancer treatment paradigms. Small molecule inhibitors that target leading or lagging strand synthesis, such as topoisomerases, DNA polymerases, DNA ligase, proliferating cell nuclear antigen (PCNA), ribonucleotide reductase, and telomerase, have been developed to clinically block uncontrolled cancer proliferation (Simon M E and Schwacha A (2014) The Mcm2-7 Replicative Helicase: A Promising Chemotherapeutic Target. BioMed Research International. vol. 2014, Article ID 549719, 14 pages, 2014). Although these compounds have demonstrated utility as chemotherapeutic agents, they are non-specific in that they target both normal and malignant DNA replication and, therefore, exhibit side effects. Unfortunately, few inhibitors that target replication initiation have been identified.
The essential role of Mcm2 in proliferation explains why Mcm2 is a useful biomarker for early detection of cancer by screening (Davies, R., Miller, R., and Coleman, N. (2005) Colorectal cancer screening: Prospects for molecular stool analysis. Nat Rev Cancer 5, 199-209). Mcm2 is expressed through all phases of the cell cycle but is lost when cells exit the cycle into quiescence, senescence, or differentiation (Stoeber, K., Tlsty, R., Happerfield, L., Thomas, G., Romanov, S., Bobrow, L., Williams, E., and Williams, G. (2001) DNA replication licensing and human cell proliferation. J Cell Sci 114, 2027-2041). Although Mcm2 binds to and detaches from DNA according to cell cycle phase, Mcm2 remains in the nucleus of higher eukaryotes throughout the cell cycle. In malignant and premalignant lesions of differentiating epithelia, there is a substantial increase in the number of cells expressing Mcm2 (Hiraiwa, A., Fujita, M., Adachi, A., Ono, H., Nagasaka, T., Matsumoto, Y., Ohashi, M., Tomita, Y., and Ishibashi, M. (1998) Specific distribution patterns of hCDC47 expression in cutaneous diseases. J Cutan Pathol 25, 285-290; Todorov, I., Werness, B., Wang, H., Buddharaju, L., Todorova, P., Slocum, H., Brooks, J., and Huberman, J. (1998) A novel proliferation marker for human tumors and normal tissues. Lab Invest 78, 73-78; Freeman, A., Morris, L., Mills, A., Stoeber, K., Laskey, R., Williams, G., and Coleman, N. (1999) Minichromosome maintenance proteins as biological markers of dysplasia and malignancy. Clin Cancer Res 5, 2121-2132). One of the characteristic features of dysplastic and malignant cells may be that they express Mcm2 (21,22).
The sensitivity of an Mcm2-based test is superior to those using other currently used markers of cell cycle entry, such as Ki67 and PCNA. Fewer cells express Ki67 than Mcm2 in malignant and dysplastic lesions. Ki67 is not expressed by all cycling cells, including cells in S phase. Furthermore, the function of Ki67 in the cell cycle remains poorly understood. PCNA shows wide variation in staining intensity in vivo. This is consistent with fluctuations in expression levels of PCNA during the cell cycle. Moreover, important roles for PCNA in DNA repair mean that PCNA is still present in non-proliferating cells and therefore less useful as a specific marker of cancer. Testing for Mcm2 represents a proven method for detecting expression of a protein at the point where growth-signaling pathways converge in the initiation of DNA replication (Gonzalez, M., Tachibana, K., Laskey, R., and Coleman, N. (2005) Control of DNA replication and its potential clinical exploitation. Nat Rev Cancer. 5, 135-141). Far less literature is available regarding the potential translational value of other replication proteins.
Approximately 4.5% of men and women will be diagnosed with colon and rectal cancer at some point in their lifetime. In 2012, there were an estimated 1,168,929 people living with colon and rectum cancer in the United States. For colon and rectum cancer, 39.5% are diagnosed at the local stage. The earlier that colon and rectal cancer is caught, the better the person has of surviving five (5) years after being diagnosed. The 5-year survival for localized colon and rectum cancer is 90.1%. It is thought to take two to three years for an asymptomatic early colorectal cancer to develop into a symptomatic advanced lesion.
There is a pressing need for new screening tests based on the increasing understanding of the biology and natural history of colorectal cancer. An effective strategy for early detection of colorectal cancer would produce very substantial benefits in overall survival. Current screening tests either detect the presence of occult blood in stool or identify gross abnormalities by endoscopy (Imperiale, T., Ransohoff, D., Itzkowitz, S., Turnbull, B., and Ross, M. (2004) For the Colorectal Cancer Study Group. Fecal DNA versus fecal occult blood for colorectal-cancer screening in an average-risk population. N Engl J Med 351, 2704-2714). Stool testing is likely to be particularly valuable, as it represents a non-invasive method for screening all of the colon and rectum without the need for bowel preparation. Methods for retrieving colon cells from stool washings are now available (Davies, R., Freeman, A., Morris, L., Bingham, S., Dilworth, S., Scott, I., Laskey, R., Miller, R., and Coleman, N. (2002) Analysis of minichromosome maintenance proteins as a novel method for detection of colorectal cancer in stool. Lancet 359, 1917-1919). However, all current tests are limited in their effectiveness and/or patient acceptability (Jackson, A., Laskey, R., and Coleman, N. (2014) Replication Proteins and Human Disease. Cold Spring Harb Perspect Biol. 6, a013060).
The replication fork helicase in eukaryotes is composed of Cdc45, the Mcm2-7 heterohexameric ATPase, and the tetrameric GINS complex (CMG assembly). The replication fork helicase (CMG) assembles in S phase in a manner that is dependent upon the replication initiation factors Sld2, Sld3, and Dpb11. Sld3 (Treslin/TICRR in humans), Sld2 (RecQL4/RecQ4 in humans), and Dpb11 (TopBP1 in humans) are required for the initiation of DNA replication, but these proteins do not travel with the replication fork. The S-phase specific kinases, cyclin-dependent kinase (CDK) and the Dbf4-dependent kinase (DDK), are also required for CMG assembly and origin activation. In late M and G1 phases, the Mcm2-7 complex loads to encircle double-stranded DNA (dsDNA) as a double hexamer. During S phase, a single-strand of DNA is extruded from the central channel of Mcm2-7, and this event is required since the CMG complex unwinds DNA by a steric exclusion mechanism.
Central to the initiation of DNA replication is the coordination of entry into S phase with origin firing. Levels of the S phase-specific kinases, S-CDK and DDK, rise during the onset of S phase, and these two kinases are central to coordinating S phase entry with origin firing. S-CDK phosphorylates Sld2 and Sld3, and these phosphorylation events are the essential functions of S-CDK (9, 10). S-CDK phosphorylation of Sld3 is conserved in human Treslin (Kumagai A, Shevchenko A, Shevchenko A, & Dunphy W (2011) Direct regulation of Treslin by cyclin-dependent kinase is essential for the onset of DNA replication. J Cell Biol. 193:995-1007). S-CDK phosphorylation of Sld2 promotes the association of Sld2 with yeast Dpb11 (Tak Y, Tanaka Y, Endo S, Kamimura Y, & Araki H (2006) A CDK-catalysed regulatory phosphorylation for formation of the DNA replication complex Sld2-Dpb11. EMBO J 25:1987-1996), and also the association of Sld2 with T-rich ssDNA (Kanter D & Kaplan D (2011) Sld2 binds to origin single-stranded DNA and stimulates DNA annealing. Nucleic Acids Res 39:2580-2592). S-CDK phosphorylation of Sld3 stimulates the association of Sld3 with Dpb11. The associations of Sld2 with Dpb11 and Sld3 with Dpb11 have been proposed to be important for the recruitment of GINS to origins, through the generation of a pre-loading complex (Pre-LC), composed of Sld2, GINS, Polε, and Dpb11 (14). S-CDK-catalyzed formation of an Sld3-Dpb11-Sld2 complex has also been proposed to be important to generate a ternary ssDNA-binding complex of high affinity, since Sld2, Sld3, and Dpb11 bind to T-rich ssDNA.
The essential role of DDK in yeast cells is the phosphorylation of subunits of the Mcm2-7 complex (Yeeles J, Deegan T, Janska A, Early A, & Diffley J (2015) Regulated eukaryotic DNA replication origin firing with purified proteins. Nature 519:431-435). DDK phosphorylation of Mcm4 is important for cell growth, and this phosphorylation event alleviates an inhibitory function of the N-terminus of Mcm4. DDK phosphorylation of Mcm4 may also promote the interaction between Cdc45 and Mcm2-7 (Sheu Y & Stillman B (2010) The Dbf4-Cdc7 kinase promotes S phase by alleviating an inhibitory activity in Mcm4. Nature 463:113-117). DDK phosphorylation of Mcm6 may also be important for cell growth (Masai H, et al. (2006) Phosphorylation of MCM4 by Cdc7 kinase facilitates its interaction with Cdc45 on the chromatin. J Biol Chem. 281:39249-39261). Mcm2 is also a target for DDK (Lei M, et al. (1997) MCM2 is a target of regulation by Cdc7-Dbf4 during the initiation of DNA synthesis. Genes Dev 11:3365-3374), and DDK phosphorylation of Mcm2 is also required for DNA replication under normal growth conditions (Bruck I & Kaplan D L (2015) The Dbf4-Cdc7 kinase promotes Mcm2-7 ring opening to allow for single-stranded DNA extrusion and helicase assembly. J. Biol Chem. 290:1210-1221). Furthermore, expression of a mutant of mcm2 (mcm2-S164A,S170A) that is not phosphorylated by DDK exerts a dominant-negative severe growth defect in budding yeast that is bypassed by the mcm5-bob1 (mcm5-P83L) mutation. The biochemical mechanism of this genetic suppression has also been examined. DDK phosphorylation of Mcm2 reduces the affinity of budding yeast Mcm2 for Mcm5, and the mcm5-bob1 mutation also reduces this affinity. This reduced affinity may help open the ‘Mcm2-Mcm5 gate,’ which may be important for the extrusion of ssDNA from the central channel of Mcm2-7 during S phase, a requirement for origin activation.
Cdc45 binds weakly to Mcm2-7 in the absence of accessory factors (Bruck I & Kaplan D (2011) GINS and Sld3 compete with one another for Mcm2-7 and Cdc45 binding. J Biol Chem 286:14157-14167). Sld3 binds tightly to Mcm2-7 and Cdc45, and thus Sld3 recruits Cdc45 to Mcm2-7 complexes. This step may further require DDK and involve the nonessential initiation factor Sld7. During origin activation, Sld3 is removed from Mcm2-7, presumably through the exposure of sequestering T-rich ssDNA. GINS can substitute for Sld3 as a factor that promotes the association of Cdc45 with Mcm2-7, thereby forming the stable Cdc45-Mcm2-7-GINS (CMG) replicative helicase complex.
The mechanism of GINS recruitment may involve the formation of the S-CDK-dependent pre-loading complex, wherein the pre-LC recruits GINS to Mcm2-7, analogous to how Sld3 recruits Cdc45 to Mcm2-7 (14). A second proposal posits that Sld3, Sld2, and Dpb11 compete with GINS for binding to Mcm2-7 prior to origin activation, blocking the premature interaction between GINS and Mcm2-7 prior to origin activation. However, when T-rich ssDNA is extruded from the central channel of Mcm2-7, an ssDNA binding surface for Sld3-Sld2-Dpb11 is generated. Sld3-Sld2-Dpb11 dissociates from Mcm2-7 once the origin is melted, since Sld3-Sld2-Dpb11 has a higher affinity for ssDNA then Mcm2-7. The dissociation of Sld3-Sld2-Dpb11 from Mcm2-7 allows GINS to bind Mcm2-7 by a passive, sequestration mechanism. The two models are not incompatible with one another, and they may both be correct.
There is an excess of Mcm2-7 double hexamer complexes loaded onto dsDNA in M phase and G1 relative to the number of Mcm2-7 double hexamer complexes that actually fire. Remarkably, the activated Mcm2-7 complexes share several features in common: DDK phosphorylation of Mcm2-7, initiation factor (Sld3, Sld2, Dpb11, Mcm10, and Sld7) binding to Mcm2-7, and Cdc45/GINS attachment to Mcm2-7. However, it is unknown what coordinates these different activities at a particular Mcm2-7 double hexamer. In other words, the mechanism that prevents Cdc45 from binding to one Mcm2-7 double hexamer, while DDK phosphorylates a different Mcm2-7 double hexamer, is unknown.
The Mcm2 protein, in particular, a component of the DNA replication apparatus, is currently being developed for its use as an early marker of colorectal cancer in cells from stool washings. Mcm2 is a subunit of the replication fork helicase, the macromolecular assembly that unwinds DNA at a replication fork.
In an initial clinical evaluation study, Mcm2-positive cells were retrieved from the stool of 37 of 40 patients with colorectal cancer, including all nine early-stage cancers, but from none of 25 control participants (Davies, R., Freeman, A., Morris, L., Bingham, S., Dilworth, S., Scott, I., Laskey, R., Miller, R., and Coleman, N. (2002) Analysis of minichromosome maintenance proteins as a novel method for detection of colorectal cancer in stool. Lancet 359, 1917-1919). Furthermore, immunohistochemistry for cell cycle proteins can address a further important challenge in diagnostic pathology, predicting the outcome of tumors using tissue sections of clinical samples. Markers that can be detected in routinely processed sections of formalin-fixed, paraffin-embedded tissue are particularly valuable. For many tumors, powerful prognostic information is provided by quantitative assessment of cell cycle entry and/or particular cell cycle phases. As accurate indicators of cell cycle state, Mcm2 has been shown to predict survival in patients with a range of tumors, including malignancies of breast (Gonzalez, M. A., Pinder, S. E., Callagy, G., Vowler, S. L., Morris, L. S., Bird, K., Bell, J. A., Laskey, R. A., and Coleman, N. (2003) Minichromosome maintenance protein 2 is a strong independent prognostic marker in breast cancer. J. Clin. Oncol. 21, 4306-4313), prostate (Meng, M. V., Grossfeld, G. D., Williams, G. H., Dilworth, S., Stoeber, K., Mulley, T. W., Weinberg, V., Carroll, P. R., and Tlsty, T. D. (2001) Minichromosome maintenance protein 2 expression in prostate: characterization and association with outcome after therapy for cancer. Clin Cancer Res 7, 2712-2718), kidney (Rodins, K., Cheale, M., Coleman, N., and Fox, S. (2002) Minichromosome maintenance protein 2 expression in normal kidney and renal cell carcinomas: Relationship to tumor dormancy and potential clinical utility. Clin Cancer Res 8, 1075-1081), bladder (Kruger, S., Thorns, C., Stocker, W., Muller-Kunert, E., Bohle, A., and Feller, A. C. (2003) Prognostic value of MCM2 immunoreactivity in stage Ti transitional cell carcinoma of the bladder. Eur Urol 43, 138-145), esophagus (Kato, H., Miyazaki, T., Fuaki, Y., Nakajima, M., Sohda, M., Takita, J., Masuda, N., Fukuchi, M., Manda, R., Ojima, H., Tsukada, K., Asao, T., and Kuwano, H. (2003) A new proliferation marker, minichromosome maintenance protein 2, is associated with tumor aggressiveness in esophogeal squamous carcinoma. J. Surg. Oncol. 84, 24-30), mouth (Kodani, I., Osaki, M., Shomori, K., Araki, K., Goto, E., Ryoke, K., and Ito, H. (2003) Minichromosome maintenance 2 expression is correlated with mode of invasion and prognosis in oral squamous cell carcinomas. J Oral Pathol Med. 32, 468-474), lung (Ramnath, N., Hernandez, F., Tan, D., Huberman, J., Natarajan, N., Beck, A., Hyland, A., Todoro, I., Brooks, J., and Bepler, G. (2001) MCM2 is an independent predictor of survival in patients with non-small-cell lung cancer. J Clin Oncol 19, 4259-4266; Hashimoto, K., Araki, K., Osaki, M., Nakamura, H., Tomita, K., Shimizu, E., and Ito, H. (2004) MCM2 and Ki-67 expression in human lung adenocarcinoma: Prognostic implications. Pathobiology 71, 193-200) and brain (harton, S., Chan, K., Anderson, J Stoeber, K., and Williams, G. (2001) Replicative Mcm2 protein as a novel proliferation marker in oligodendrogliomas and its relationship to Ki67 labelling index, histological grade and prognosis. Neuropathol Appl Neurobiol 27, 305-313; Hunt, D., Freeman, A., Morris, L., Burnet, N., Bird, K., Davies, T., Laskey, R., and Coleman, N. (2002) Early recurrence of benign meningioma correlates with expression of mini-chromosome maintenance-2 protein. Br J Neurosurg. 16, 10-15; Scott, I., Morris, L., Rushbrook, S., Bird, K., Vowler, S., Burne, N., and Coleman, N. (2005) Immunohistochemical estimation of cell cycle entry and phase distribution in astrocytomas: Applications in diagnostic neuropathology. Neuropathol Appl Neurobiol 31, 455-466). Moreover, for the data sets analyzed, the Mcm2 labeling index was also superior to clinico-pathological parameters that are currently widely used to predict outcome, namely, histological grade and lymph node stage. Furthermore, DDK, which phosphorylates Mcm2 at serine 53, is overexpressed in colorectal cancer cells (Bonte, D., Lindvall, C., Liu, H., Dykema, K., Furge, K., and Weinreich, M. (2008) Cdc7-Dbf4 kinase overexpression in multiple cancers and tumor cell lines is correlated with p53 inactivation. Neoplasia 10, 920-931).
It was found that in budding yeast, a model organism for DNA replication, Mcm2 is phosphorylated by DDK in a manner that is stimulated by the replication initiation protein Sld3 (Bruck, I., and Kaplan, D. (2015) The replication initiation protein Sld3/Treslin orchestrates the assembly of the replication fork helicase during S phase. J Biol Chem 290, 27414-27424). DDK phosphorylates serine 170 of Mcm2, and this single phosphorylation event is essential for the initiation of DNA replication (Hua, C., Zhao, G., Li, Y., and Bie, L. (2014) Minichromosome Maintenance (MCM) Family as potential diagnostic and prognostic tumor markers for human gliomas. BMC Cancer 14, 526; Toschi, L., and Bravo, R. (1988) Changes in cyclin/proliferating cell nuclear antigen distribution during DNA repair synthesis. J Cell Biol 107, 1623-1628). DDK phosphorylation of Mcm2 is essential because this phosphorylation event opens the helicase ring, allowing single-stranded DNA to be melted at an origin of replication (Hua, C., Zhao, G., Li, Y., and Bie, L. (2014) Minichromosome Maintenance (MCM) Family as potential diagnostic and prognostic tumor markers for human gliomas. BMC Cancer 14, 526). Melting of origin DNA is essential because it allows for the genomic DNA to be subsequently unwound by the helicase during DNA replication (Hua, C., Zhao, G., Li, Y., and Bie, L. (2014) Minichromosome Maintenance (MCM) Family as potential diagnostic and prognostic tumor markers for human gliomas. BMC Cancer 14, 526).
It was also found that in human cells, the analogous phosphorylation reaction occurs, and DDK phosphorylates human Mcm2 at serine 53 (Bruck, I., and Kaplan, D. (2015) The replication initiation protein Sld3/Treslin orchestrates the assembly of the replication fork helicase during S phase. J Biol Chem 290, 27414-27424). The human homolog of Sld3, called Treslin, stimulates this phosphorylation event (Id.). Thus, DDK phosphorylation of Mcm2 is a fundamental event in the cell cycle that triggers the initiation of DNA replication (Id.). This reaction is conserved from yeast to humans (Id.). For these reasons, probing for DDK-phosphorylated Mcm2, instead of the traditional Mcm2, is potentially a more specific assay for detecting early cancer. Whereas the current tumor marker Mcm2 is present at equal levels throughout the cell cycle, phosphorylated Mcm2 is present at substantially higher levels during S phase, when DNA replication is active (Hua, C., Zhao, G., Li, Y., and Bie, L. (2014) Minichromosome Maintenance (MCM) Family as potential diagnostic and prognostic tumor markers for human gliomas. BMC Cancer 14, 526). Since cancer cells spend a higher fraction of time in S phase compared to normal cells, cancer cells will likely have higher levels of DDK-phosphorylated-Mcm2 compared to normal cells (Davies, R., Miller, R., and Coleman, N. (2005) Colorectal cancer screening: Prospects for molecular stool analysis. Nat Rev Cancer 5, 199-209). In addition, colorectal cancer cells have higher levels of DDK than normal cells, suggesting that Mcm2-S53P will be abundant in colorectal cancer cells (Bonte, D., Lindvall, C., Liu, H., Dykema, K., Furge, K., and Weinreich, M. (2008) Cdc7-Dbf4 kinase overexpression in multiple cancers and tumor cell lines is correlated with p53 inactivation. Neoplasia 10, 920-931).
The sensitivity of an Mcm2-based test is superior to those using conventional markers of cell cycle entry, such as Ki67 and PCNA. Ki67 is not expressed by all cycling cells, including cells in S phase. Moreover, important roles for PCNA in DNA repair mean that PCNA is still present in non-proliferating cells and therefore less useful as a specific marker of cancer. Testing for Mcm2 is a proven method for detecting expression of a DNA replication protein. However, Mcm2 lacks specificity, because it is present in all stages of the cell cycle.
Accordingly, what is needed is a more directed, effective screening assay based on an antibody specific for a phosphorylated Mcm2. However, in view of the art considered as a whole at the time the present invention was made, it was not obvious to those of ordinary skill in the field of this invention how the shortcomings of the prior art could be overcome.
While certain aspects of conventional technologies have been discussed to facilitate disclosure of the invention, Applicants in no way disclaim these technical aspects, and it is contemplated that the claimed invention may encompass one or more of the conventional technical aspects discussed herein.
The present invention may address one or more of the problems and deficiencies of the prior art discussed above. However, it is contemplated that the invention may prove useful in addressing other problems and deficiencies in a number of technical areas. Therefore, the claimed invention should not necessarily be construed as limited to addressing any of the particular problems or deficiencies discussed herein.
In this specification, where a document, act or item of knowledge is referred to or discussed, this reference or discussion is not an admission that the document, act or item of knowledge or any combination thereof was at the priority date, publicly available, known to the public, part of common general knowledge, or otherwise constitutes prior art under the applicable statutory provisions; or is known to be relevant to an attempt to solve any problem with which this specification is concerned.